Integration of reverse transcribed human immunodeficiency virus type 1 (HIV-1) cDNA into a chromosome of an infected cell is essential for virus replication. Thus, the HIV-1 integrase (IN) enzyme and integration step are attractive targets for developing antiviral drugs against HIV/AIDS. Understanding the detailed mechanism of HIV-1 integration in vivo is critical for effective anti-IN and anti-integration drug development. Integration in vivo is mediated by large nucleoprotein preintegration complexes (PICs) that form following viral infection and reverse transcription, and PICs isolated from infected cells can integrate their endogenous cDNA copy into an added target DNA in vitro. The parent R01 grant focuses on the structure and function of HIV-1 PICs. In this proposal two different innovative technologies that were unavailable when the parent grant was written will be used to significantly enhance the pace and quality of research. Recently described real-time quantitative (RQ)-PCR assays for quantifying levels of in vivo integration as well as the in vitro integration activity of PICs will drive the experimental design. The RQ-PCR PIC integration assay will increase the sensitivity of detecting in vitro integration approximately 10,000-fold over the currently used Southern blotting assay, as well as reduce the time required for results from approximately one week to one day. This will significantly impact the ability of the research to move directly and rapidly toward addressing the central issues which include determining the roles of host protein cofactors in HIV-1 integration as well as critical details of IN protein-protein and protein-DNA interactions required for integration in vivo. Additionally, small-interfering RNA, a previously unavailable technique for knocking-down the expression of genes in mammalian cell lines, will be used alongside RQ-PCR to determine the roles of previously-implicated host protein factors in HIV-1 integration in vivo. The results will determine the roles of host factors in HIV-1 integration, as well as protein-protein and protein-DNA interactions within PICs essential for HIV-1 integration in vivo. These results will significantly add to the understanding of HIV-1 integration as it occurs in infected cells, which will in turn aid the discovery and development of antiviral drugs targeted against HIV-1 IN and integration.